Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P<0.05), but not significantly in the liver and duodenum (P>0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (P<0.05) compared with healthy birds. Furthermore, the infection reduced absorption of orally administered enrofloxacin, significantly decreased Cmax  and AUC0-12h  of enrofloxacin, but increased Tmax , T1/2a and V/F . Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers.P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of  Colibacillosis is a common disease in poultry, affecting the growth of broilers and also the physiological function of the small intestine in the chickens. Fluoroquinolones have been successfully used to treat colibacillosis in poultry, among which enrofloxacin is most frequently applied abcb1 gene, and belongs to ATP-binding cassette (ABC) superfamily, which is involved in drug transport In vitro and in vivo studies have demonstrated that P-gp can modulate the pharmacokinetics of human medicines, resulting in drug-drug interactions of structurally diverse compounds E. coli infection.P-glycoprotein (P-gp) is encoded by E. coli infection on the expression levels of P-gp and CYP3A  in the liver, kidney and small intestine to clarify whether different expression levels of P-gp and CYP3A affect intestinal absorption, biliary secretion and kidney excretion of enrofloxacin in broilers. Furthermore, we investigated the influence of verapamil, an inhibitor of P-gp, on the absorption of enrofloxacin. The results indicate that E. coli infection modulates the pharmacokinetics of orally administered enrofloxacin by increasing intestinal P-gp expression and decreasing CYP3A expression in the liver and kidney of broilers.The present study examined the effects of Ross 308 broilers  were purchased from a local commercial poultry farm . All birds were kept at 25°C, had free access to standard commercial feed (without additives) and water, and treated following the protocol approved by Nanjing Agricultural University Animal Care and Use Committee. Before starting the experiment at the age of 4-week old, all broilers were verified free from colibacillosis. Mouse monoclonal anti-P-gp (C219) antibody, used for immunohistochemistry (IHC), was from Covance . Rabbit anti-mouse IgG-horseradish peroxidase (HRP) was purchased from Boster . Verapamil was purchased from Sigma  and enrofloxacin was bought from China Institute of Veterinary Drug Control. All other reagents were purchased commercially with highest quality.Escherichia coli (E. coli) O2 strain, isolated from the broilers with colibacillosis, was kindly provided by the Laboratory of the Microbiology, Nanjing Agricultural University. The strain was stored at −80°C prior to use. The day before infection, E. coli was inoculated onto LB agar and incubated at 37°C for 24 h. Then eight colonies were suspended in 10 ml of broth and incubated at 37°C for 6 h when its OD600 value was about 0.6, as quantified by ultraviolet spectrophotometry. At the age of 4-week old, each broiler was given 0.5 ml of the overnight culture containing 1.5×108 colony forming units (cfu) by pectoral muscle injection. Some broilers exhibited typical clinical signs of colibacillosis, including respiratory stress, and white loose droppings within 12 h after inoculation. Tissue samples collected from the dead broilers at necropsy were then cultured to confirm that the strain used to inoculate the birds caused the mortalities. In this study, three groups of broilers were infected with E. coli, including one group for immunohistochemistry of P-gp and mRNA quantitation (abcb1), and two groups for pharmacokinetic analysis of enrofloxacin.E. coli infected (n = 5) broilers. Total RNA was isolated from individual tissues of all birds using Trizol Reagent  according to the manufacturer's instructions. All RNA samples were treated with 100 U DNase I  for 30 min at 37°C to ensure that the samples were free of genomic DNA contamination. The total RNA concentration was then quantified using a Nanodrop photometer . Ratios of the optical density (OD) values at 260/280 nm of all preparations were between 1.8 and 2.0. Each RNA sample was subjected to electrophoresis on a 1.4% agarose formaldehyde gel to verify its integrity. Single-stranded cDNAs were synthesized and real time PCR was performed, as described previously abcb1, CYP3A and β-actin were designed as described −ΔΔCt method Real time RT-PCR was used to detect the Abcb1 and CYP3A mRNA expression levels in the liver, kidney and different parts of small intestine in healthy (n = 5) and E. coli infected broilers at 4-week old, immunohistochemical staining was performed as described previously E. coli infected broilers were collected. The tissue-sections were prepared and incubated overnight at 4°C with the primary antibody  and at 37°C for 1 h with the secondary antibody (rabbit anti-mouse IgG-HRP), and the P-gp immunoreactivity was visualized with DAB staining. Sections without incubation with the primary antibody served as negative controls. Three semi-quantitative measurements for P-glycoprotein staining were performed by two experimental pathologists in a double-blind analysis under a light microscope  equipped with Plan Apo objectives connected to a CCD camera  as previously described To study the localization and semi-quantitative protein expression of P-gp in the liver and small intestine of healthy and E. coli challenge. Group IV, 30 min prior to enrofloxacin (10 mg/kg b. w.) oral administration, was treated with verapamil (15 mg/kg b. w.) orally following 12 h of E. coli challenge. Plasma samples for HPLC analysis were drawn in heparin tubes 20 min before and at 0.083, 0.25, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8 and 12 h following enrofloxacin administration in each group. The samples were placed on ice before transporting to the laboratory and centrifuged at 1 500 g for 10 min. The centrifuged plasma was harvested and aliquot for storage at −80°C before HPLC analysis.Forty 28-day old broilers were randomly divided into 4 groups with 10 broilers in each group in this study. Group I birds received enrofloxacin orally with single dose of 10 mg/kg b.w. through crop tube gavages. Group II was pre-treated with verapamil (15 mg/kg b.w.) orally for 30 min, followed by oral administration of enrofloxacin with single dose of 10 mg/kg b.w as previously reported 18 HPLC Columns . The flow rate of the mobile phase was set to 0.85 ml/min. UV absorbance was measured at 278 nm.The plasma concentrations of enrofloxacin were detected through Agilent 1200 high-performance liquid chromatography (HPLC) system as described previously with minor modification 0-12h) was calculated according to the linear trapezoidal method.Pharmacokinetic calculations were performed on each individual set of data using 3p97 practical pharmacokinetic software . The best fit of compartment model was determined according to the Akaike’s Information Criterion. The area under the concentration–time curve  test for individual comparisons. Values of mRNA abundance were expressed as the fold change relative to the average value of one group. Pharmacokinetic parameters of enrofloxacin were analyzed using student E. coli culture containing 1.5×108 cfu/ml by pectoral muscle injection. We found that within 12 h of inoculation, two broilers died of the infection and others showed clinical signs at different degrees. Some broilers exhibited typical clinical signs of colibacillosis, including respiratory stress, and white loose droppings. Necropsy lesions showed a layer of white cellulose pseudo-membranous covering the surface of the liver and heart. Also, severe bleeding and swelling were observed in most segments of the small intestine except duodenum. E. coli was also isolated in liver from the diseased birds.For colibacillosis model, each broiler was inoculated with 0.5 ml of abcb1 and CYP 3A37 were detected by real time PCR with β-actin chosen as a housekeeping gene for normalization. Compared with the healthy group, the abcb1 mRNA level in the kidney and small intestines was up-regulated after E. coli infection. As shown in abcb1 mRNA was detected at significantly higher levels in kidney (P = 0.032), jejunum (P = 0.017) and ileum (P = 0.046) of infected birds than that of healthy broilers, whereas the mRNA levels in duodenum (P = 0.349) and liver (P = 0.264) were not significantly changed in E. coli infected broilers, though an increasing trend was observed. In different tissues of infected broilers, the highest level of abcb1 mRNA was observed in ileum, and followed by jejunum, liver, kidney and duodenum . Positive staining was detected in liver, kidney, jejunum and ileum of healthy and E. coli infected birds as shown in E. coli infected broilers. Similarly in kidney, the main staining was observed in the apical plasma membranes of proximal tubule cells of healthy broilers, but a punctuate labeling for P-gp was distributed widely throughout the cytoplasm in the kidneys of infected broilers. To validate our immunohistochemical results, we semi-quantified the stained liver, kidneys and small intestines from healthy and E. coli infected broilers using Image-Pro Plus 4.1 software. For each experimental group, IOD, positive area and score for 25 samples from each bird was analyzed, and a comparison was made between the average values obtained (E. coli infection treatment significantly enhanced (P<0.05) P-gp staining of jejunum and ileum via IOD, positive area and scores evaluation. Though the total P-gp level was increased in liver and kidney, the level in the bile canalicular membranes and apical plasma membranes of proximal tubule cells was not significantly changed via IOD, positive area and score estimates. In kidney, the area of positive staining was enhanced, but IOD and score were not changed. Quantification of P-gp staining in intestine provided additional validity to our studies at protein level, which was coincident with the trend of changes in mRNA expression changes between healthy and infected birds.To date, there are no reports on the effects of obtained . Comparevs time curves for enrofloxacin are shown in E. coli are presented in maxC (P = 0.000) and 0-12hAUC  as well as obviously longer maxT (P = 0.040)and 1/2aT (P = 0.045), indicating that the absorption of enrofloxacin was slower and inhibited. Consistent with these findings, higher mRNA expression of P-gp was observed in the small intestine in broilers after E. coli infection. However, infected birds demonstrated lowered Cl/F (P = 0.038) but higher 1/2eT (P = 0.000) and volume of distribution (P = 0.040), compared with healthy birds, suggesting that the clearance of enrofloxacin was also inhibited by E. coli infection.To further confirm whether P-gp and CYP3A expression levels are the main factors affecting the pharmacokinectics of enrofloxacin in broilers after infection, both infected and healthy broilers were administered a single dose of enrofloxacin orally. Following oral administration, a one-compartment open model was found to best fit concentration-time data of enrofloxacin. The plasma concentration E. coli infection modulates the pharmacokinetics of oral enrofloxacin by targeting the intestinal P-glycoprotein expression, the pharmacokinetics of enrofloxacin was also studied after exposure to verapamil (a potent and selective inhibitor of P-gp inhibitor) for 30 min in both healthy and infected groups. The plasma concentration of enrofloxacin in the presence or absence of oral verapamil (15 mg/kg b.w.) was measured after oral administration of enrofloxacin (10 mg/kg). The plasma concentration-time profiles are shown in max (1.46- and 2.94-fold) and AUC 0-12h (1.33-fold and 2.39-fold) of enrofloxacin increased in healthy and infected broilers, respectively. Meanwhile, the Tmax of enrofloxacin was significantly short and Ka of enrofloxacin was significantly higher when P-gp was inhibited with verapamil. The plasma concentration of enrofloxacin was altered in both healthy and E. coli infected broilers, which was prevented by verapamil, indicating that the absorption was P-gp-dependent.To investigate whether E. coli infection affects the pharmacokinetic parameters of enrofloxacin by modifying P-gp expression levels in the tissues of 4-week old broilers. The main finding of the present study is that E. coli infection resulted in an increased expression of abcb1 and P-gp in the small intestine and kidney. The observed changes in the expression level of P-gp mRNA/protein suggest that E. coli infection affected P-gp expression at the transcriptional and translation levels. Likely, E. coli infection-induced change in the absorption of enrofloxacin was due to increased expression of P-gp in the small intestine. This is also supported by the data that decreased Cmax and AUC0-12h of enrofloxacin in E. coli infected broilers was paralleled to increased abcb1 mRNA and P-gp protein in jejunum and ileum. The decrease in plasma level of enrofloxacin was especially evident shortly after enrofloxacin administration, suggesting that the intestinal uptake was inhibited. Though the absorption of enrofloxacin declined, volume of distribution increased and terminal half-life became longer, which might be associated with decreased CYP450 level or attenuated function of P-gp in liver and kidney, leading to decreased clearance of enrofloxacin in infected birds compared with healthy ones.In this study, we focused on clarifying whether abcb1 and CYP450 are rapidly and significantly repressed E. coli infection up-regulated P-gp expression in broilers, which is much different from that in mammals where LPS induced down-regulation of P-gp during the acute phase response via nuclear receptors E. coli bacteria); E. coli may induce more cytokines mediating more cell signaling pathways. It has been pointed out that LPS is not a generic substitute for bacterial infection to study its effect on P-gp expression, function and on drug pharmacokinetics E. coli infection or LPS on the P-gp expression in poultry. Clearly, further research is required to address this issue. Nevertheless, our results are in line with other findings bAbcb1mRNA and protein in mammary epithelial monolayer and BME-UV cells as well as in mice, and Shiga-like toxin II increases P-gp expression in mouse brain Injection of bacterial lipopolysaccharide (LPS) is a widely used model of inflammation and is well characterized regarding its effect on P-gp expression. LPS-medicated induction of pro-inflammatory cytokines activates a variety of cell signaling molecules, including c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) abcb1 by E. coli infection following therapy was likely to reflect a beneficial effect on the bird, as this up-regulation might improve the barrier function of the gastro-intestinal tract through effluxing toxins, including LPS, and enhance the resistance against further infections by viruses and bacteria E. coli infection also reduced absorption of orally administered enrofloxacin. Therefore, when antimicrobial agents are used in clinics to treat E. coli infections, both of the above two aspects should be taken into consideration in order to reach effective concentrations of the drugs in animal body.The increase in the levels of abcb1 mRNA in liver and kidney in infected broilers based on the longer half-life time and volume of distribution . The present experiments were designed to test the hypothesis that loss of P-gp transport activity of bile canaliculus and tubule is accompanied by a shift in transporter protein from the luminal membrane to a compartment where it can no longer function as an efflux transporter. Therefore, we further detected the expression of P-gp and its localization using IHC method. The images showed that P-gp was internalized into the cytoplasm away from the biliary membrane in the liver and apical plasma membranes of proximal tubule in the kidney of E. coli infected broilers but not in healthy birds (showed by E. coli infection might have adversely affected protein trafficking as P-gp went to the cell surface or led to mislocalization of P-gp on the plasma membrane, resulting in a reduced capacity to transport substrate. The result was similar to the finding that LPS can induce the translocation of MRP2 from the canalicular membrane into cytoplasmic membrane vesicles by failure to anchor properly the membrane protein to the cytoskeleton, resulting in mislocalization in the cell membrane in vitro and in vivo is posttranslational and dependent on protein phosphorylation-based signaling E. coli infection.We found that the efflux rate of enrofloxacin through bile canalicular and tubular secretion was not paralleled to up-regulation trend of E. coli infection increased mRNA and P-gp expression in the intestine, but decreased CYP3A expression in the liver and kidney in broilers. Consequently, this modulated the pharmacokinetics of enrofloxacin in broilers. The findings provide strong evidence that it is essential to optimize the usage and dosage of enrofloxacin in E. coli infected broilers for a better treatment of colibacillosis. Also, from the present study, we should be aware that a healthy animal model cannot substitute for an infection animal model to evaluate the pharmacokinetics of enrofloxacin in broilers.In summary, here, for the first time, we showed that


Candida albicans is a complex, layered network of rigid structural polysaccharides composed of β-glucans and chitin that is covered with a fibrillar matrix of highly glycosylated mannoproteins. Poly-morphonuclear cells  are the most prevalent circulating phagocytic leukocyte in peripheral blood and they are pivotal in the clearance of invading fungal cells from tissues. The importance of cell-wall mannans for the recognition and uptake of C. albicans by human PMNs was therefore investigated. N- and O-glycosylation-deficient mutants were attenuated in binding and phagocytosis by PMNs and this was associated with reduced killing of C. albicans yeast cells. No differences were found in the production of the respiratory burst enzyme myeloperoxidase (MPO) and the neutrophil chemokine IL-8 in PMNs exposed to control and glycosylation-deficient C. albicans strains. Thus, the significant decrease in killing of glycan-deficient C. albicans strains by PMNs is a consequence of a marked reduction in phagocytosis rather than changes in the release of inflammatory mediators by PMNs.The cell wall of the opportunistic human fungal pathogen,  C. albicans is ordinarily found as a commensal yeast colonizing the human gastrointestinal tract. It is, however, able to invade and cause deep-seated infections in individuals with compromised host defenses arising from trauma, the administration of immunosuppressive agents, a variety of disease and association with a number of genetic mutations that affect immune recognition 2 core structure (numbers in subscript indicating repeating units), appended to more structurally diverse outer chains - mannose backbone to which are attached side chains consisting of α--β-- and α-- linked mannose and phosphoman-nose branches r chains . In C. abranches . C. albill types -30.C. albicans O-linked mannans are simpler structures, consisting of a linear (unbranched) chain of mannose residues, predominantly α--linked -linked  26,31].,31.C. althe wall . C. albiirulence  and adheirulence ,34, dispirulence .C. albicans cell wall N- and O-linked glycosylation on the fungus-human PMN interaction, we used a series of C. albicans strains defective in different cell wall glycosylation components. The host-fungus interaction was characterized by quantifying the proportions of C. albicans bound or engulfed by the PMNs, the percentage of fungal cells killed following co-incubation, and PMN activation by measurement of supernatant myeloperoxidase and IL-8. Additionally, the sensitivity of C. albicans glycosylation-defective strains to PMN-derived anti-microbial compounds was investigated.To investigate the importance of 600 reached 0.6. The cells were then washed three times in sterile phosphate-buffered saline , and resuspended to a final concentration of 1 × 108 cells/ ml in PBS. Prior to use, an aliquot of the required number of cells was labeled with 0.1 mg/ml fluorescein 5-iso-thiocyanate  in 0.05 M carbonate-bicarbonate buffer , for 15 min at room temperature with continual agitation. Cells were then washed three times with sterile PBS and used immediately in experiments with C. albicans cells.All strains used in this study are listed in g to generate platelet-poor plasma (PPP). The lymphocyte and Ficoll-Paque PLUS layers were aspirated and discarded. The pellet, containing erythro-cytes and PMNs was lysed using RBC lysis buffer  according to the manufacturer's instructions. The resulting PMN pellet was washed twice in sterile PBS at room temperature, with centrifugation at 400 g and finally resuspended in pre-warmed RPMI-1640 medium supplemented with 10% donor's PPP. Neutro-phils were examined by light microscopy and cell viability was established as >98% by Trypan Blue dye exclusion [Human PMNs were isolated from freshly isolated, EDTA-anticoagulated peripheral venous blood of healthy human volunteers . Brieflyxclusion . All tes6 PMNs were labeled with anti-CD13.PE-Cy5  according to the manufacturer's instructions for 30 min at room temperature in the dark. After two washes in sterile PBS with centrifugation at 400 g the cells were ready for use.As required aliquots of 1 × 106 washed FITC-labeled C. albicans cells were co-incubated with 1 × 106 freshly isolated, anti-CD13. PE-Cy5 labeled human PMNs (effector: target ratio 3:1), and co-incubated in RPMI-1640 medium supplemented with 10% donor's own serum for 30 min at 37°C with continuous shaking. Cells were harvested by centrifugation at 500 g, resuspended and fixed in 500 μl ice-cold PBS with 40 mM EDTA and 1% (v/v) paraformaldehyde, and stored on ice.A sample of 3 × 106 FITC-labeled C. albicans cells were co-incubated with 1 × 106 freshly isolated, anti-CD13. PE-Cy5 labeled human PMNs (effector: target ratio 3:1), and co-incubated in RPMI-1640 medium supplemented with 10% donor serum for 150 min at 37°C with continuous shaking. No hypha formation was observed inside the neutrophils by this time, thus no killing of neutrophils occurred due to hypha formation within the phagolyso-some. Fungal cells were harvested by centrifugation at 1500 g, and the supernatant discarded. Hypotonic lysis of the PMNs  was followed by three washes in sterile PBS. Finally the released C. albicans cells were resuspended in 1% paraformaldehyde in PBS, containing 4 μg/ml propidium iodide (PI). The percentages of dead cells with PI-stained nuclei were quantified by fluorescence microscopy. A minimum of 3 groups of 100 cells were counted for each strain and the experiment repeated for a total of six independent biological replicates. Controls in the absence of PMNs showed 0% killing, therefore all killing was ascribed to the activities of the PMNs. For fluorescence images on PMNs with attached and phagocytosed C. albicans cells a Zeiss Axioplan 2 microscope (63 × magnification) with the Hamanatsu C4742-95 digital camera was used and images were analyzed with Openlab software V4.0.4.Samples of 3 × 10C. albicans cells. In principle, internalized fungal cells are encapsulated by the intact cell membrane of the PMN, thereby preventing their staining by CFW. Free, suspended C. albicans cells or cells adhered to the outside of the neutrophil are stained by the fluorophore and were detected via the Pacific Blue channel.Flow cytometry analysis of the samples was carried out using a LSRII flow cytometer , with computer-assisted evaluation of the generated data by FACS Diva software . The fungal cell wall dye Calcofluor White (CFW) was added to a final concentration of 10 μg/ml immediately prior to flow cytometry, allowing distinction between bound and engulfed C. albicans cells were assayed for markers of PMN activation. Simultaneous screening of 36 cytokines, chemokines and other secretory proteins was carried out using a human cytokine profiler kit  according to the manufacturer's instructions. The cytokine profiler data, in addition to conventional sandwich ELISA-based analysis confirmed evidence from other groups showing that activated neu-trophils release the respiratory burst associated proteins myeloperoxidase and lactotransferrin (LTF) into the environment when stimulated [Supernatants collected following co-incubation of PMNs and imulated . IL-8 seC. albicans. Incubations were carried out for 30 min to evaluate glycosylation effects on adherence and phagocytosis. In the following description, we take 'association' to mean all Candida cells co-sorting with PMNs following the incubation period (including adhered and phagocytosed subsets), whilst 'attached' or 'adhered' cells refer to subsets of the associated cells bound to the PMN cell surface, undergoing phagocytosis. Various subsets of C. albicans yeast cells that were interacting with PMNs were distinguished in the analysis. An 'association' indicates C. albicans cells that co-sort with PMNs and includes adhered and phagocytosed yeast cells. Yeast cells that were 'attached' or 'adhered' refers to subsets of associated cells that were bound to the PMN cell surface but were not phagocytosed while 'engulfed' refers to C. albicans cells that had been phagocytosed. The strains used in this study are shown in och1Δ strain lacks the branched, outer part of the N-glycan structure [pmr1 Δ strain has a truncated O-glycan and N-glycan structure [mnt1Δ/mnt2Δ strain contains only a single O-linked mannose residue [mnn4Δ strain lacks cell wall phosphomannan including the acid-labile β-1,2 mannan fraction [C. albicans serotype B strain A9 differs from the serotype A strains in being deficient in β-1,2 mannose in the acid stable component of the N-linked mannan [To characterize the effects of changing glycosylation status upon host: fungus interaction, we challenged purified human PMNs with a range of glycosylation-defective strains of tructure , the pmrtructure , the mnt residue  and the fraction . C. albid mannan . SerotypC. albicans by neutrophils was also validated by fluorescence microscopy. Two sub-populations of C. albicans cells could be distinguished using FACS . C. albicans cells in quadrant Q1-2  represent engulfed cells whilst those in quadrant Q2-2  represent cells attached to the PMN surface : Materials and Methods. After 30 min co-incubation, approximately 36 ± 2%  of the control C. albicans cells were co-sorted with PMNs  suggesting that O-mannan or β 1,2 linked mannan was implicated in PMN recognition and binding. PMN cells were attenuated in their ability to engulf N- and O-glycosylation-defective C. albicans strains   strain to 64 ± 6% (och1Δ), 60 ± 3% (pmr1Δ), and 62 ± 4% (mnt1Δ/mnt2Δ) for the glycosylation-defective mutants respectively. Complementation of the deleted genes in all cases reverted the ratio of engulfed:adhered C. albicans to that resembling the control strain. The mnn4Δ strain lacking N-linked cell wall phosphomannan exhibited a relatively minor reduction in engulfment (53 ± 3%) in comparison to the other strains tested, although this was still significantly less than the control strain  but not changes in the β 1,2 mannose content, reduced the engulfment of adherent fungal cells by human PMNs.Flow cytometry was used to investigate the adherence and engulfment of ith PMNs . The mnt strains . Analysi < 0.01) . Consequl strain . Likewisild type . The C. trophils . TherefoC. albicans phagocytosis by purified anti CD13-PE-CY5-labeled human PMNs was validated independently by microscopy. PMNs were co-incubated with FITC-labeled C. albicans glycosylation mutants. After fixation, the C. albicans cell wall dye, Calcofluor White, was added immediately prior to microscopy to distinguish between C. albicans cells adhered to the PMN surface and those that had been internalized through phagocytosis. Prolonged exposure to the fixation solution  resulted in non-specific staining of the PMN nuclei by Calcofluor White, presumably due to increased permeability of the cell membrane. Therefore, to ensure that any non-specific staining did not interfere with FACS analysis, samples were analyzed minutes after fixation when no nonspecific CFW staining was observed. The majority of the CAI4-CIp10 (wild type control strain) cells associated with PMNs fluoresced only under the FITC filter, suggesting that they had been phagocytosed and were not exposed on the PMN surface. However, the C. albicans strains that were defective in cell wall glycosylation fluoresced under both the FITC and Pacific Blue channels, indicating that the majority of C. albicans cells had adhered to the PMN surface, but that the PMNs were unable to phagocytose the cells. Reintroduction of a single copy of the desired gene again restored the ability of the PMNs to phagocytose the C. albicans strain to almost wild type levels, confirming that the glycosylation status of C. albicans is important for phagocytosis  .Next we assessed whether changes in the fungal cell-wall glycosylation status altered susceptibility to killing by PMNs. The sensitivity of s period . Glycosy = 0.15) . ComplemC. albicans by several mechanisms and reciprocally the ability of C. albicans to reduce PMN viability may be affected by the presence of other microorganisms and antigens. PMNs can ingest and digest pathogens but also release of large quantities of enzymes and toxic free-radicals into the environment, causing damage and death to non-ingested pathogens in the vicinity of the immune cells [C. albicans.PMNs can kill ne cells . Here weC. albicans glycosylation-deficient strain on PMN viability by Trypan Blue exclusion. Stimulation by LPS, heat-killed Pseudomonas aeruginosa and heat-killed Escherichia coli resulted in a higher proportion of dead PMNs , as compared to pre-stimulation with any of the C. albicans strains CAI-4 (28 ± 3%),pmr1Δ (29 ± 1%) and pmr1 Δ + PMR1 (37 ± 7%) respectively . Therefore, more PMNs were killed following co-incubation with bacterial cells and LPS as compared to following co-incubation with the C. albicans strains tested.First, we investigated the effect of 30 min co-incubation with LPS, two bacterial and one C. albicans mutant strains would affect the composition and quantity of microbicidal compounds released into the cell-culture supernatant . The results in SI Table S1 indicate that following the 30 min pre-incubation period, fungal cells generally elicited higher levels (20-25 ng/ml) of MPO production by PMNs as compared to LPS (9 ± 1 ng/ml), P. aeruginosa (13 ± 2 ng/ml) and E. coli (12 ± 4 ng/ml). IL-8 induction was similar irrespective of the stimulus (∼16 ± 2 ng/ml) C. albicans was characterized by co-incubation with fungal cells over 150 min.Next, we investigated whether PMNs exposure to wild-type and C. albicans cells following 150 min incubation with cell culture supernatants from pre-stimu-lated PMNs showed no statistically significant difference, with respect to the pre-stimulant. All the C. albicans strains tested in this assay were equally sensitive to supernatant from pre-stimulated PMNs  . Supernatant concentrations of MPO and IL-8 did not change during co-incubation with fungal cells (SI Table S1 - online only). Yeast strains of the pmr1Δ mutant showed similar sensitivity to cell damaging agents released by activated neutrophils when compared to their wildtype control.The percentage of dead C. albicans strains was characterized by quantifying supernatant levels of secreted markers of activation. Initial testing using the R&D Systems proteome profiler cytokine assay kit , which has been effective in the analysis of cytokine release from monocytes [Activated PMNs are able to secrete a variety of cytokines and chemokines into their immediate environment, resulting in autoinduction and the generation of chemoattrac-tant gradients. The activation of PMNs in response to stimulation by glycosylation-defective onocytes  and T-ceonocytes , identifC. albicans strains (LTF was not studied in detail as similar to MPO it is a marker of the respiratory burst). MPO catalyses the formation of hypochlorous acid and other toxic reactive oxidants following neutrophil activation, and has been shown to play a key role in the control of C. albicans infection by PMNs [PMN cell activation was characterized by investigating the production of myeloperoxidase (MPO) and the cytokine IL-8 (CXCL8) in cell culture supernatant following 30 min co-incubation with  by PMNs ,42,43. I by PMNs ,45. TherC. albicans resulted in a basal level of MPO production (16 ± 2 ng/ml). Co-incubation with C. albicans resulted in increased levels of MPO with a mean of 22 ± 5 ng/ml. There was no significant difference in the induction of MPO production between the glycosylation-deficient and parent strains of C. albicans tested . However, no significant differences in IL-8 production were found between the mutant and wildtype strains that were tested. Thus PMNs upon exposure to C. albicans produced MPO and IL-8, but this was not dependent on the glycosy-lation status of the cell wall.Analysis of the cell culture supernatant of PMNs that had not been exposed to s tested . FollowiC. albicans is the most common cause of invasive noso-comial disease [C. albicans cell wall glycosylation on the binding and phagocytosis by PMNs. We show that O-glycosylation plays a role in the initial association between PMNs and Candida as O-glycan-deficiency resulted in significantly fewer cells associated with PMNs as compared to control and N-glycosylation mutant strains. In addition, loss of cell wall N- and O-glycans results in a reduced capacity of PMNs to phagocytose fungal cells. PMNs were able to phago-cytose significantly fewer och1Δ, pmr1Δ, and mnt1Δ/ mnt2Δ mutant cells as compared to the control strain. Binding of these mutants was not abolished however, implying that residual cell wall components such as β-glucans also play a role in uptake by PMNs. In such strains a corresponding increase in the percentage of adhered cells was found reflecting the disruption of the engulfment process leading to an accumulation of C. albicans on the PMN cell surface. The data suggest that recognition and uptake of fungal cells by PMNs is a two-step process and suggests that independent receptor-ligand interaction mediated by cell surface mannans may be required, for each stage to be successfully completed. This is in keeping with previous observations for the phagocytosis of apoptotic cells where a two step process for successful phagocytosis has been proposed [ disease ,47. Incr disease  and thos disease  highlighproposed .C. albicans virulence in a murine model [N- and O-glycan-deficient mutants as compared to the mnn4Δ strain indicating the relatively minor role of phosphomannan as compared to glycosylation, in the recognition, attachment and phagocytosis by neutrophils. Therefore non-phagocytosis-dependent interactions and adhesion to PMNs is unlikely to be due to charge interactions with PMN in the C. albicans cell wall. This highlights important differences in the phagocytosis of C. albicans between macrophages and PMNs as recent data shows that loss of phosphomannan content profoundly reduces the ability of macrophages to recognize and ingest C. albicans whereas loss of O-mannan enhances phagocytosis [Candida albicans adhered to and phagocytosed by PMNs was not significantly different in the serotype A and serotype B strains. The serotype B strain bears cell wall mannans containing β1, 2 mannose residues, recently shown to be associated with both N and O mannans [Previous work from our laboratory has shown that loss of cell wall phosphomannan had little impact upon ne model . This stocytosis . Loss ofocytosis ,51. The  mannans . This suC. albicans by PMN cells upon specific receptor-ligand combinations have been described previously. Lavigne et al. showed that fungal cell wall β-glucan is important for the recognition of adhered C. albicans cells, and is able to stimulate the antimicrobial respiratory burst in PMNs [C. albicans cell surface was described by Yamamura et al., who showed that complement factor C3 was required for C. albicans killing, but not phagocytosis [et al. links these two studies by showing that murine dectin-1 -deficient neutrophils were found to be impaired in their ability to bind un-opsonized zymosan particles, resulting in a lowered respiratory burst [C. albicans cells restored binding, however the dectin-1 deficient PMNs remained attenuated in their ability to generate the respiratory burst. Dectin-1 deficiency resulted in a strong decrease in the ability of PMNs to kill unopsonized C. albicans cells, however, killing was restored with opsonized C. albicans [C. albicans cell wall mannan has also been shown to inhibit the interaction between C. albicans cells and human PMNs, demonstrating the requirement for glycans during attachment, and supports the data from our study [C. albicans showed that TLR2 expression on neutrophils was required for efficient recognition of and chemotaxis towards fungal cells. Additionally, neutrophils from TLR2-/- mice were shown to be attenuated in their phagocytic activity towards C. albicans cells as well as expressing lower levels of nitric oxide and myeloperoxidase [The dependence of the processes of recognition, phagocytosis and killing of  in PMNs . The impocytosis . Work byry burst . The usealbicans . Purifieur study . Studiesroxidase .C. albicans glycosylation mutants tested in our study. Our data confirmed that contact with fungal cells results in activation-dependent secretion of MPO and IL-8 into the culture medium [C. albicans cells with cell culture supernatant from pre-stimulated PMNs showed that: (i) there was no significant difference between the levels of MPO and IL-8 induced by the control strain and the pmr1Δ glycosylation-deficient mutant; and (ii) there was no significant difference in the percentage of C. albicans killed between the control and glycosylation-deficient strain. These data together suggest that PMNs at least in terms of their secretory profile do not respond specifically to the cell wall glycans of C. albicans cells, instead generating a broad non-specific cellular response to the fungus.There were no significant differences in the levels of MPO or IL-8 produced by PMNs in the first 30 min following contact with the e medium ,42,58,59N- and O-linked glycans are involved in the recognition, binding and phagocytosis of C. albicans by human PMNs. Interestingly, studies using human peripheral blood mono-nuclear cells have found that N-linked glycans are predominantly recognized by the mannose receptor whilst O-glycans bound TLR4 and that these interactions are important for cytokine-stimulating activity by yeast cells [This study is the first to show that ubiquitous cell wall st cells . Here we


Staphylococcus aureus in the nasopharynx, body surface and vagina. Most invasive infections by this bacterium are endogenous, caused by strains spread from the nasopharynx of carriers. S. aureus is a pathogen involved in the etiology of hospital- and community-acquired infections. Transplant and dialysis patients are at risk of colonization or infection by multi-resistant S. aureus. Infection is directly linked to individual immunity, and the major histocompatibility complex (MHC) plays a crucial role in determining susceptibility to diseases. Different MHC specificities have been shown to be more frequent in individuals suffering from certain diseases. This study aimed to investigate the association between HLA class I (HLA-A and -B) and class II (HLA-DRB1) molecules and nasal carriage of S. aureus in dialysis and kidney transplant patients at a hospital in Southern Brazil.Healthy individuals can host ® SSO (One Lambda). Nasal swab samples of S. aureus were isolated from the nasal cavity (both nostrils) of patients undergoing dialysis or kidney transplantation.The sample consisted of 70 dialysis and 46 kidney transplant patients, totaling 116 patients. All subjects were typed for HLA molecules using LABTypeS. aureus. Allele A*68 was not observed in the carrier group, but the allele was observed six times in the non-carrier group (p = 0.0097). Regarding HLA-B and HLA-DRB1, no allele was shown to be involved in protection against or susceptibility to carriage of S. aureus. In kidney transplant patients, allele A*03 was more frequent in the non-carrier (20.83%) than in the carrier (5.88%) group (p = 0.0486). HLA-B*15 was present in carriers (5.88%) and non-carriers (25%) (p = 0.0179). Regarding class II alleles, DRB1*03 appeared to be related to susceptibility to carriage of S. aureus (p = 0.0319).In renal dialysis patients, HLA-A*02 was the most frequent allele in both carriers (25.5%) and non-carriers (21.2%) of S. aureus in transplant patients. In addition, HLA-A*68  and HLA-A*03 and HLA-B*15 (transplant patients) appear to be associated with increased resistance to S. aureus nasal carriage.Our findings suggest that HLA-DRB1*03 may be involved in susceptibility to nasal carriage of  Staphylococcus aureus is considered an important etiologic agent due to its high frequency and pathogenicity, causing diseases in both healthy and immunocompromised individuals. This microorganism is able to spread easily in the hospital environment and to develop resistance to antimicrobial agents [l agents ,2.S. aureus in the nasopharynx, another 30% carry it intermittently, and 50% are not carriers [S. aureus are at higher risk of developing infection [Approximately 20% of adults are carriers of carriers . Persistnfection . Dialysinfection .Infection is directly related to individual immunity and the major histocompatibility complex (MHC)--a set of genes found on the short arm of human chromosome 6--plays a crucial role in determining susceptibility to diseases. Specifically, MHC contains a group of genes that code for several proteins expressed on the surface of a variety of cell types. In humans, these molecules are known as human leukocyte antigens (HLA) .+). Class II molecules are distributed less widely, being found primarily on the surface of cells linked directly to the immune response , with the purpose of presenting peptides to regulatory T cells (CD4+). The class III region does not encode histocompatibility molecules, but rather other molecules such as tumor necrosis factors, C4 and C2 proteins, complement factor B, heat shock protein, and 21-hydroxylase enzyme [Molecules of the HLA system are classified as class I , class II , and class III. These molecules are highly polymorphic glycoproteins that differentiate themselves from one another by their location in tissues and their function. Class I molecules are present on the surface of all nucleated cells and are responsible for presenting peptides to cytotoxic T cells  and class II (HLA-DRB1) molecules and nasal carriage of The study population consisted of 70 renal patients undergoing dialysis and 46 kidney transplant patients, totaling 116 patients. All subjects were typed for HLA class I  and class II (DRB1) molecules and evaluated for nasal carriage. Biological material, nasal discharge and blood were collected between June and November 2009.S. aureus were isolated from the nasal cavity of patients undergoing dialysis or kidney transplantation by inserting sterile swabs into both nostrils. The material was placed in Petri dishes (90 × 15 mm) containing mannitol salt agar (MSA) , transferred to a test tube containing trypticase soy broth (TSB) with 6.5% NaCl, and then placed in a stove for 24 hours. After 24-48 hours of incubation at 37°C, suspected S. aureus colonies were subjected to Gram staining and immersion microscopy. Those identified as Gram-positive cocci arranged in grape-like clusters were transferred to a TSB medium with 6.5% NaCl. The tube coagulase test was performed after six hours of incubation. After the identification tests, samples were stored in a TSB medium with glycerol (20%) and frozen at -20°C for later analysis.Nasal swab samples of Human DNA extraction: Peripheral blood was collected by venipuncture in vacuum tubes containing EDTA anticoagulant, and genomic DNA was extracted using EZ-DNA reagent, according to the manufacturer's instructions .® SSO kit was used in combination with the Luminex™ technology for typing of HLA class I (HLA-A and -B) and class II (HLA-DRB1) alleles.Genetic polymorphism of HLA molecules: One Lambda LABTypeS. aureus. The number of times a given allele was found (n) and allele frequency (Af) were quantified in both patient groups. P-value was calculated using Fisher's exact test . Bonferroni correction  was used to calculate p values below 0.05. The odds ratio (OR) and confidence interval (95%CI) were calculated whenever the p value was lower than 0.05.For data analysis, dialysis and transplant patients were subdivided into carriers and non-carriers of The study was approved by the Research Ethics Committee of Universidade Estadual de Maringá (protocol no. 212/2009), and written informed consent was obtained from all participants. The study was conducted in accordance with the provisions of the Declaration of Helsinki.S. aureus and 33 (47.1%) were non-carriers.Of 70 dialysis patients, 30 (42.8%) were female and 40 (57.1%) male. Age ranged from 22 to 85 years. Thirty-seven patients (52.8%) were nasal carriers of S. aureus and 12 (26%) were non-carriers.Of 46 patients evaluated in the transplant group, 16 (34.7%) were female and 30 (65.2%) male. Age ranged from 18 to 64 years. Thirty-four patients (74%) were nasal carriers of p = 0.0097). This result suggests a tendency towards protection or resistance against nasal carriage of S. aureus. Regarding HLA-B and -DRB1, no allele was shown to be involved in protection against or susceptibility to carriage of S. aureus.Analysis of the frequency of class I, A and B alleles, and class II, DRB1 alleles, (Table p = 0.0486), which suggests a tendency towards protection against carriage of S. aureus. HLA-B*15 also showed a trend towards protection against carriage  (p = 0.0179). Regarding class II alleles, DRB1*03 appeared to be related to susceptibility to carriage of S. aureus (p = 0.0319).As shown in Table S. aureus in dialysis and kidney transplant patients.The molecules of the HLA system, which are directly linked to the immune response, play a crucial role in the susceptibility to or protection against infectious diseases . The detS. aureus [S. aureus, making individuals more susceptible to carrying the bacterium. Class I (HLA-Bw35) and class II (HLA-DR2 and HLA-DR1) antigens were also shown to be involved in resistance against non-carriage of the bacterium.During the literature review, we found only one study on HLA and nasal carriage of . aureus , which eS. aureus. It also contributed to demonstrate the involvement of HLA class I specificities  as protective factors against nasal carriage of S. aureus in renal patients.The present study contributed to confirm the involvement of HLA-DRB1*03  as a possible genetic marker for susceptibility to nasal carriage of S. aureus, in particular those with a multi-resistant profile, lies in the fact that this pathogen is linked to endogenous infections [The importance of identifying patients carrying fections . In thatfections .S. aureus in transplant patients. In addition, HLA-A*68  and HLA-A*03 and HLA-B*15 (transplant patients) appear to be associated with increased resistance to S. aureus nasal carriage.Our findings suggest that HLA-DRB1*03 may be involved in susceptibility to nasal carriage of S. aureus. Establishing the role of host genetic factors should contribute to a better understanding of infectious conditions as well as to develop new therapies according to the progress in the field of pharmacogenetics.Further studies are warranted to clarify the role of HLA as a genetic marker in resistance against or susceptibility to carriage of The data set supporting the results of this article is available in the repository of Universidade Estadual de Maringá (UEM), at UEM Digital Library, under no. VTLS 000184756/www.bce.uem.br.The authors declare that they have no competing interests.SDB carried out HLA typing, drafted the manuscript and revised it critically for the intellectual content. RRS participated in the collection of biological material, processing and analysis of data. JB participated in the design of the study, helped to draft the manuscript and revised it critically for the intellectual content. WVSJ participated in the design of the study and performed the statistical analysis. LBG participated in the collection of biological material, processing and analysis of data, helped to draft the manuscript and revised it critically for the intellectual content. All authors read and approved the final manuscript.


Caenorhabditis elegans specifically responds to infection by the up-regulation of certain genes, distinct pathogens trigger the expression of a common set of genes. We applied new methods to conduct a comprehensive and comparative study of the transcriptional response of C. elegans to bacterial and fungal infection. Using tiling arrays and/or RNA-sequencing, we have characterized the genome-wide transcriptional changes that underlie the host's response to infection by three bacterial  and two fungal pathogens (Drechmeria coniospora and Harposporium sp.). We developed a flexible tool, the WormBase Converter (available at http://wormbasemanager.sourceforge.net/), to allow cross-study comparisons. The new data sets provided more extensive lists of differentially regulated genes than previous studies. Annotation analysis confirmed that genes commonly up-regulated by bacterial infections are related to stress responses. We found substantial overlaps between the genes regulated upon intestinal infection by the bacterial pathogens and Harposporium, and between those regulated by Harposporium and D. coniospora, which infects the epidermis. Among the fungus-regulated genes, there was a significant bias towards genes that are evolving rapidly and potentially encode small proteins. The results obtained using new methods reveal that the response to infection in C. elegans is determined by the nature of the pathogen, the site of infection and the physiological imbalance provoked by infection. They form the basis for future functional dissection of innate immune signaling. Finally, we also propose alternative methods to identify differentially regulated genes that take into account the greater variability in lowly expressed genes.While  C. elegans is now widely used as a model organism to dissect host innate immune responses to bacterial, fungal and viral pathogens C. elegans that accompany infection by different pathogens, often using microarrays, have identified many genes involved in the response to infection pmk-1, the MAP2K gene sek-1 and the MAP3K gene nsy-1 are crucial in both the intestine and the epidermis to counter infection. Only in the intestine, however, does activation of the pathway involve the protein kinase D gene dkf-2cnc antimicrobial peptide genes, it does so through divergent pathways sek-1 and nsy-1, pmk-1 does not have a role in neuronal development C. elegans and used tiling arrays and/or RNA-seq to characterize the transcriptional response of C. elegans to three bacterial and two fungal pathogens. These techniques have allowed us to define in far greater detail transcriptional responses to infection. We developed tools to allow the comparison of this new data to results from previous studies. These will be available to the research community, and will have a general utility for any study involving large numbers of genes. Using this tool, we were able to generate high-confidence lists of biomarkers. We have also defined groups of genes and functional annotation categories that are shared in the transcriptional response to more than one pathogen and groups of genes that are specifically regulated in response to only one pathogen, thereby directly contributing to the fuller characterization of innate immune responses in C. elegans.The complete understanding of this complexity requires comprehensively cataloguing the gene expression changes that accompany infection. In this study we have extended our previous analyses of the innate immune response of C. elegans by the fungus D. coniospora or a number of different bacterial pathogens, using cDNA- and/or oligo-arrays S. marcescens, E. faecalis or P. luminescens were 0.90, 0.88, 0.91 and 0.92, respectively) consistent with similar comparisons We previously characterized the transcriptional changes in host genes that accompany infection of S. marcescens, E. faecalis and P. luminescens all colonize the intestine of C. elegans. Infection with each of the bacteria caused the expression of between 2,000 and 3,000 genes to be up-regulated . Otherwise, there was a common decrease in the expression of ribosomal genes and genes involved in protein translation and transcription  was isolated from an infected C. elegans individual found in a rotting apple collected in Orsay (Essonne), France, in October 2008. It is therefore likely to be a natural pathogen of C. elegans.In addition to the bacterial data sets, we also collected RNA-seq data from samples of worms infected with two fungal species, C. elegans infected with D. coniospora and Harposporium, we found that close to 1,500 and 2,000 genes were up-regulated, respectively, and similar numbers of genes were down-regulated  and dop-1 (dopamine receptor). We also found dgk-1, whose activity regulates locomotion, too. In addition, we identified 3 of the nematode's globin-like genes, some of which are known to be expressed in the sensory neurons and linked to oxygen sensing, which is an integral part of the detection of food, as well as eat-18, a member of the gustatory class of receptors, and daf-28, which can be considered as a starvation response gene One relatively frequent theme among the 272 genes that were down-regulated by the two fungal pathogens  was a liS. marcescens, E. faecalis and P. luminescens among the genes induced by both D. coniospora and Harposporium, and of genes up-regulated upon infection by each of the bacterial pathogens among the genes down-regulated upon infection with both fungal pathogens  is much greater than the number of genes induced both by the three bacteria and the epidermal pathogen D. coniospora (43) . Additionally, in both the Harposporium- and the bacterially-induced genes there was an enrichment of genes identified as intestinally expressed, based on mRNA tagging Harposporium and the three bacteria establish an intestinal infection, as opposed to D. coniospora, which infects via the epidermis. The genes induced by intestinal pathogen-infection included genes with known involvement in the response to intestinal infection, e.g. eight C-type lectins and the lysozyme lys-2. In addition, we found the trypsin-like proteases try-3 and try-7, which have not yet attracted attention as putative defense effectors, as well as the ERK MAPK gene mpk-2, which as noted above is a candidate regulator of the intestinal response  . There wresponse . Gene caby dbl-1 . Furtherhis-8 . This difference in relative frequencies was still apparent when we defined five equal classes of peptides based on their length . ClearlyC. elegans are absent from the plant pathogenic nematode Meloidogyne incognitaCaenorhabditis, detailed locus-specific studies have shown that there are marked differences for some categories of defense genes, including the inducible nlp and cnc genes, which have undergone multiple duplications in C. elegans compared to C. remanei or C. brenneriC. briggsae, C. remanei, C. brenneri, C. japonica, Pristionchus pacificus, Brugia malayi, and Trichinella spiralis. These transcripts correspond to 3,421 C. elegans genes, or 18.4% of the total set of genes detected by RNA-seq in uninfected worms. When we calculated the percentage of genes conserved among the genes up-regulated after infection with any of the five pathogens, we found that it was significantly lower . Although these figures cannot be entirely trusted, since the current genome sequences of the other nematode species are not complete, this is further evidence for the unusually rapid evolution of defense genes.When comparisons are made across a broad range of host species, effectors of innate immunity are often found to be highly divergent, probably as a result of strong selective pressure. For example, many gene classes important in C. elegans, the interpretation of our previous studies was based on gene models and functional annotations present in the nematode-specific database WormBase at the time http://wormbasemanager.sourceforge.net/), that enables the conversion of lists of genes with different identifiers and from different WormBase versions to a single coherent format, using the WB ID, referenced to a defined version of WormBase. The WB IDs are linked to the physical coordinates in the genomes, as are microarray probes and RNAi clones, so that these automatically follow evolutions in gene structure. If a gene has changed, the output list given by the Wormbase Converter will contain the new name  and thereby block their action. Of the 27 members in the fip and nlp genes after infection with any one of the three bacterial intestinal pathogens. Since some intestinal genes like mtl-1 and -2 are also down-regulated upon bacterial infection, this cannot explain all observed transcriptional changes. Indeed, there are now examples of pathogens triggering down-regulation of host genes . But from a biological perspective, it is not at all obvious that change in absolute transcript number is a meaningful measure when looking across large sets of genes with an enormous range of expression levels. For example, one can imagine a case where there are 1000 transcripts of gene X under condition A, and 1100 under condition B; for gene Y there are 10 and 110, respectively. In both cases the change in absolute number is the same (100), but for gene X, the relative fold change is 1.1 and for gene Y it is ten-fold higher. One might expect the change in Y to have more biological impact.Alternative methods have been developed but these all use either the Poisson distribution or Fisher's exact test to model data, neither of which cope well with biological variation abf-4, cnc-5, cnc-9, fipr-8, fipr-23, ins-8, ins-32 and ins-39 and several nematode-specific peptides. Remarkably, only 4 of the 619 genes figure in the list of genes identified by oligo-array as being induced by D. coniospora infection (data not shown). Methods based on differences in transcript counts would allow these genes to be identified, otherwise, another way to address this issue is to set the value for the condition with no expression to some arbitrary small value We therefore experimented with two alternative approaches see . InspectC. elegans genes that are involved in the transcriptional response to infection. Extending previous observations, we have shown that not only is the host response composed of both pathogen-specific and pathogen-shared elements, but that the latter depends upon the site of infection. To enable this work, we needed to construct bioinformatic tools, which we expect to be of broad utility to the research community, especially as with the advent of improved sequencing methods, the predicted structures of genes are constantly evolving.This study has greatly expanded our understanding of the molecular repertoire of the P. luminescens strain Hb was cultured in liquid LB medium at 30°C overnight, spread on NGM plates, incubated for 24 h at 30°C, then another 20 h at 25°C. E. faecalis strain OG1RF was cultured in liquid BHI at 37°C overnight, spread on NGM plates, incubated for 24 h at 37°C, then cultured another 20 h at 25°C. OP50 was cultured in liquid LB medium at 37°C overnight, spread on NGM plates, incubated for 24 h at 37°C, then cultured another 20 h at 25°C. S. marcescens strain Db10 fer-15 for fungal infections). Synchronized L1 larvae were transferred to NGM agar plates spread with E. coli strain OP50. N2 worms were cultivated at 25°C until the young adult stage (43–45 h) and then transferred to plates spread with S. marcescens, E. faecalis or P. luminescens and cultivated at 25°C for 24 h. For D. coniospora and Harposporium sp. infection, fer-15 worms were cultivated at 25°C until the L4 stage (36 to 38 hours) before being exposed to fungal spores. Nematodes were harvested after 12 hours for D. coniospora and 24 hours for Harposporium sp. This time-point was chosen for Harposporium sp. as the infection proceeded at roughly half the rate as seen with D. coniospora . Worms were harvested and washed twice in M9. As far as possible, larvae were separated from adults during washing. The volume of the pellet was measured and 10 volumes of TRIZOL (Gibco BRL) added. Tubes were vortexed for 45 sec, placed in liquid Nitrogen, flash frozen completely, then thawed completely in a 37°C water-bath. This freeze/thaw process was repeated 4 times. The homogenate of worms and Trizol was frozen at −80°C and sent to the Reinke laboratory  on dry ice.Eggs were allowed to hatch in the absence of food at 25°C  or 15°C  but had not published is given in S. marcescens data, for genes with expression >40 PSL in data sets I and II D. coniospora data, lists of up- and down-regulated genes were taken from For RNA-seq and tiling arrays, log2 fold changes between gene expression values of infected versus uninfected nematodes were calculated. For log2 fold changes >0.00001 the values >81.25th percentile were defined as up-regulated and for log2 fold changes <−0.00001 the values <18.75th percentile were defined as down-regulated. For oligo-arrays, lists of up- and down-regulated genes were taken from C. elegans genes do not mention the WormBase release used for data annotation, we included the possibility of identifying the most likely WormBase release for a given input list of genes. Lastly, published gene lists often contain a heterogeneous mix of gene identifiers; WormBase Converter can handle such lists and also displays any gene name for which an associated WB ID gene identifier cannot be found. Furthermore, if a new WormBase version is released, the tool automatically retrieves the new data from the server this making it easy to keep lists up to date. WormBase Converter runs under most operating systems .Changes in gene annotation and gene structure occur between different versions of WormBase. This hampers a precise comparison of data sets obtained using different WormBase versions for annotation. The WormBase Converter addresses this problem by maintaining a continuously up-dated list of all annotation changes, using the standard WB ID. Any input list of genes, using other gene identifiers (e.g. sequence or transcript names) is first converted to a list of WB ID gene identifiers, using a correspondence table from the appropriate WormBase release. The tool then takes into account all changes in gene annotation that occurred between different versions of WormBase  and modifies the corresponding WB ID gene identifiers in the list of genes. As many researchers are not familiar with WB ID gene identifiers, but know public gene names, or sequence names, these formats can be chosen for the output list. Furthermore, all changes that occurred during the conversion are displayed in a list to enable tracking of genes that are changed or deleted in the output release. To overcome the fact that many publications that include lists of http://wormbasemanager.sourceforge.net/.We used the freely available software package EASE http://www.pangloss.com/seidel/Protocols/venn.cgi) and the Venn diagram plotter (http://ncrr.pnl.gov/software/ ) were used to create Venn diagrams and proportional Venn diagrams, respectively.Graphpad Prism 5 was used for creating figures and statistical analysis. The Venn diagram generator  from the line joining the 99th percentiles can be calculated, giving an estimate of the degree to which a given gene represents an outlier in the data. Equivalent calculations can also be performed to identify down-regulated genes.For both approaches, the expression values are first log10 transformed. The first method that is described in the legend to www.cisred.org) and converted to genes (WS170) using the WormBase Converter. All transcripts with expression in the RNA-seq data set were also converted to genes (WS170). The chi-square test was used to compare the numbers of up-regulated genes after infection with the different pathogens that are conserved to the percentage of conserved genes among the expressed genes.A set of transcripts (WS170) conserved between nematode species was retrieved from the cisRED database , genomic coordinates chromosome III: 7711900 to7717600, showing gene models, obsolete gene models, RNAi clones and microarray probes according to WS222. The gene C03B8.1 has undergone a merge with C03B8.3. The microarray probe cea2.i.18533 was associated with the gene C03B8.1 until WS216 but is associated with gene C03B8.3 from WS217 on.(PDF)Click here for additional data file.Figure S2S. marcescens infection.Transcripts differentially expressed after  Expression of transcripts in uninfected (x-axis) versus S. marcescens infected (y-axis) worms. Dot plot showing log10 transformed dcpm values obtained by RNA-seq. (A) Transcripts up- or down-regulated in tiling arrays, RNA-seq and cDNA-arrays are highlighted in red or green, respectively. (B) Transcripts up- or down-regulated in tiling arrays and RNA-seq are highlighted in red or green, respectively. (C) Transcripts up-regulated in tiling arrays and RNA-seq with low expression but high induction are highlighted in red.(PDF)Click here for additional data file.Figure S3Alternative approach of defining up- and down-regulated transcripts. (A) Dot plot showing log10 dcpm values for expression of transcripts in uninfected worms (x-axis) versus D. coniospora infected worms (y-axis). (B) Dot plot after 45 degree clockwise rotation. The rectangles separate the bins and define upper and lower y limits to each bin. The central x value on the limit y line is marked with a small square. (C) A hyperbole is then fitted to these small squares. Transcripts whose data points lie outside of the hyperbole are defined as up-regulated (red) or down-regulated (blue).(PDF)Click here for additional data file.Figure S4Overlap of transcripts defined as up-regulated by three methods. Dot plots showing log10 transformed dcpm values for expression of transcripts in uninfected (x-axis) versus D. coniospora infected worms (y-axis). Transcripts identified as up-regulated using three approaches Click here for additional data file.Table S1S. marcescens for 24 hours using tiling arrays.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns Db10 and OP50 are the log2 transformed ratios of the perfect-match to mismatch probe intensities, mapped on to individual transcripts Click here for additional data file.Table S2S. marcescens for 24 hours using RNA-seq.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns Db10 and OP50 are the dcpm values calculated for each individual transcript Click here for additional data file.Table S3E. faecalis for 24 hours using tiling arrays.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns EF and OP50 are the log2 transformed ratios of the perfect-match to mismatch probe intensities, mapped on to individual transcripts Click here for additional data file.Table S4E. faecalis for 24 hours using RNA-seq.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns EF and OP50 are the dcpm values calculated for each individual transcript Click here for additional data file.Table S5P. luminescens for 24 hours using tiling arrays.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns PL and OP50 are the log2 transformed ratios of the perfect-match to mismatch probe intensities, mapped on to individual transcripts Click here for additional data file.Table S6P. luminescens for 24 hours using RNA-seq.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns PL and OP50 are the dcpm values calculated for each individual transcript Click here for additional data file.Table S7D. coniospora for 12 hours using RNA-seq.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns DC and OP50 are the dcpm values calculated for each individual transcript Click here for additional data file.Table S8Harposporium sp. for 24 hours using RNA-seq.Expression of transcripts after infection with  Public gene, sequence and transcript names come from WS170. The values in the columns Harposporium and OP50 are the dcpm values calculated for each individual transcript Click here for additional data file.Table S9Lists of differentially regulated genes. Lists of up- or down-regulated genes (WS210) after infection with different pathogens, established by RNA-seq (A), tiling arrays (B), oligo-arrays (C) and cDNA-arrays (D).(XLS)Click here for additional data file.Table S10Genes regulated by bacterial infection. Lists of genes up-regulated by infection with bacterial pathogens (RNA-seq) (A). Genes up-regulated by infection with all three bacterial pathogens (RNA-seq) (B). Lists of genes down-regulated by infection with bacterial pathogens (RNA-seq) (C). Genes down-regulated by infection with all three bacterial pathogens (RNA-seq) (D). Gene lists are based on WS210.(XLS)Click here for additional data file.Table S11Gene categories regulated by bacterial infection. Shared gene categories up- (A) or down-regulated (B) upon bacterial infection (RNA-seq).(XLS)Click here for additional data file.Table S12Genes regulated by bacterial and/or fungal infection. Lists of genes up- (A) or down-regulated (B) by infection with fungal and bacterial pathogens (RNA-seq). Genes up-regulated by infection with two fungal and three bacterial pathogens (RNA-seq) (C), with Harposporium sp. and D. coniospora (RNA-seq) (D), with Harposporium sp. and three bacterial pathogens (RNA-seq) (E). Genes down-regulated by infection with Harposporium sp. and D. coniospora (RNA-seq) (F), with Harposporium sp. and three bacteria (RNA-seq) (G). Gene lists are based on WS210.(XLS)Click here for additional data file.Table S13Gene categories regulated by bacterial and/or fungal infection. Shared gene categories up (A) and down-regulated (B) upon fungal and bacterial infection (RNA-seq).(XLS)Click here for additional data file.Table S14Overlap between platforms of genes up-regulated after infection. Numbers of C. elegans genes detected as up-regulated after S. marcescens (A), P. luminescens (B), E. faecalis (C) and D. coniospora (D) infection. The bold values along the diagonal indicate the total number of genes classed as up-regulated using the different experimental techniques. The off-diagonal values indicate the numbers or percentages of genes out of each total that were also found using the two techniques. For each technique, the number of genes included in the analysis (see ysis see  is shown(PDF)Click here for additional data file.Table S15Genes regulated by S. marcescens infection. Genes up-regulated after S. marcescens infection: genes shared between data sets (A). Potential biomarkers for S. marcescens infection (B). Gene lists are based on WS210.(XLS)Click here for additional data file.Table S16E. faecalis or P. luminescens infection.Genes regulated by  Genes up-regulated after E. faecalis (A) or P. luminescens (B) infection: genes shared between data sets. Gene lists are based on WS210.(XLS)Click here for additional data file.Table S17D. coniospora for 12 hours using oligo-arrays.Expression of genes after infection with  The table was generated from previously unpublished data, publicly available at Array Express (E-MEXP-479). Median ratio of gene expression in infected versus uninfected nematodes (A). Genes up- or down-regulated after D. coniospora infection (B). CDS sequence names are based on WS150.(XLS)Click here for additional data file.Table S18D. coniospora infection.Genes regulated by  Genes (WS210) up-regulated after D. coniospora infection (12 h): genes shared between data sets.(XLS)Click here for additional data file.


Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms.There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent NaE. coli K12 and E. coli BL21 pRSET eYFP-IL6 in mineral media with glucose and glycerol as carbon (C) sources, respectively. With glucose as the C-source it was possible to reduce the required buffer concentration in shake flask cultures by 50%. Moreover, with glycerol as the C-source, no buffer was needed at all.The controlled-release discs presented here were successfully used to cultivate These novel polymer-based controlled-release discs allowed buffer concentrations in shake flask media to be substantially reduced or omitted, while the pH remains in the physiological range of the microorganisms during the whole cultivation time. Therefore, the controlled-release discs allow a better control of the pH, than merely using high buffer concentrations. The conditions applied here, i.e. with significantly reduced buffer concentrations, enhance the comparability of the culture conditions used in screening experiments and large-scale fermentation processes. Shaken bioreactors are the most important reaction vessels for screening production strains and for process development. Therefore, several thousand shake flask experiments are performed each year ,2.Escherichia coli is approximately 0.3 Osmol/L, increasing or decreasing the osmolarity results in reduced bacterial growth rates  per consumed molecule of ammonium [E. coli produce acetate. This also decreases the pH-value. Once the glucose is depleted and the pH in the medium is not too low for growth of the microorganisms, the accumulated acetate is consumed as a second carbon source and the pH subsequently increases [The pH-value is an important parameter for cultivating microorganisms. The best pH-range for cultivating e.g. perature ,13. The ammonium , the pH ncreases ,18. Morencreases . Therefoncreases .One of the most important differences between small- and large-scale is the control of the pH of the medium. Weuster-Botz et al. presented a system for pH-control in shake flasks that applies pH-probes, pumps, storage vessels for pH controlling agents and other equipment ,20. HoweEscherichia coli K12 and Escherichia coli BL21 pRSET eYFP-IL6 were used as model microorganisms. The strain E. coli BL21 pRSET eYFP-IL6 produces a fusion protein of enhanced Yellow Fluorescent Protein (YFP) and interleukin-6. The two strains were cultivated in media containing glycerol or glucose as carbon source, respectively.An easy-to-use polymer-based controlled-release system for keeping the pH in shake flasks in a reasonable range is presented in this paper. It is based on the fed-batch system described by Jeude et al. . This neE. coli K12 and E. coli BL21 pRSET eYFP-IL6 [E. coli BL21 pRSET eYFP-IL6 cultures additionally contained 100 μg/mL ampicilin.E. coli cultivations [4)2SO4; 0.5 g/L NH4Cl; 3 g/L K2HPO4; 2 g/L Na2SO4; 0.5 g/L MgSO4·7H2O; 41.85 g/L (0.2 M) 3-(N-morpholino)-propanesulfonic acid (MOPS); 0.01 g/L thiamine hydrochloride; 1 mL/L trace element solution . For cultivations with E. coli BL21 pRSET eYFP-IL6 0.1 g/L ampicillin were added. The pH was adjusted to 7.5 with NaOH. The typical MOPS buffer concentration of 0.2 M was used for precultures and for reference cultivations with glucose and glycerol, respectively. For other experiments different MOPS buffer concentrations and different initial pH-values were used and are mentioned in the respective experiment description.Modified Wilms & Reuss synthetic medium (henceforth referred to as Wilms-MOPS medium) was used for ivations ,22. The For online monitoring of oxygen transfer rates (OTR) of all cultures a Respiration Activity Monitoring System (RAMOS) device, fabricated in-house and previously described by Anderlei et al. ,24, was 2CO3 was developed. In this work disc-shaped controlled-release systems were used. The controlled-release systems are produced as foils during the manufacturing process. Then discs are cut out from the foil. These deliver the preferred mostly linear release kinetics of the sodium carbonate.To keep the pH-value of a cultivation in a narrow range without using high buffer concentrations, a polymer-based controlled-release system with embedded Na2CO3 was supplied by Sigma Aldrich . The Na2CO3 was milled with a vibration micromill  in a high-grade steel mortar and then sieved through test sieves . The fraction with particle sizes ranging from 20 to 50 μm was used. First, a mixture consisting of component A of Sylgard™184, Na2CO3 (30% (w/w)) and catalyst was weighed and degassed in a desiccator in a 30 mbar vacuum for 0.5 h. Then, component B was added. The finished mixture was subsequently casted as a thin foil onto a glass plate with a casting knife (gap 1.1 mm) and then cross-linked at 50°C in a convection oven for 3 h. Then discs having a diameter of 15 mm were stamped out (surface area 405.26 mm2) and applied for the experiments. The disc-shaped controlled-release systems then gradually release the embedded Na2CO3 in an aqueous system at a pre-determined rate, so that the medium was alkalized during cultivation, thus counteracting any biological acidification.The release system was composed of commercially available solvent-free two-component  silicone Sylgard™184 as well as the catalyst Syl-off™ 4000  in a concentration of 0.1% (w/w). The platinum catalyst Syl-off™ 4000 accelerates the cross-linking of the two components. The ratio between the two components A and B was 10:1 as recommended by the manufacturer. Analytical grade NaThe controlled-release discs can not be autoclaved, as the wet environment dissolves the sodium carbonate from the matrix. For the presented experiments the controlled-release discs were sterilized with 70% ethanol and, subsequently, washed with sterile water for one minute. This sterilization method was used directly before the experiments starts. For long term sterilization the controlled-release discs can be treated with gamma rays.2CO3 from the controlled-release discs, an Na+-ion selective electrode was used. With this electrode it is generally possible to determine the concentration of Na+-ions within the range from 10-6 mol/L to 1 mol/L. The electrode consists of two half cells, a reference electrode (inLab Reference Pro) and the electrode for the Na+-ions. The measuring instrument (SevenMulti) and the electrodes were purchased from MettlerToledo . All salts used were purchased in the highest purity from Sigma Aldrich . Destilled water was prepared in a Millipore unit . It was used for the preparation of the solutions and kept at a temperature of 20°C. As bridge electrolyte inside the Na+-ion selective electrode a 0.1 mol/L NH4Cl-solution was applied. As ion adjustment buffer (ISA) an NH4Cl/NH3-solution was prepared by adding 200 g NH4Cl to 50 ml concentrated NH3-solution and the volume was filled up with water to a final value of 1 L. For the measurement, the destilled water with ISA-buffer was adjusted to a pH-value of 7 with 1 M KOH. As master solution for the calibration and to store the Na+-ion selective half cell, a 0.1 M NaCl solution was prepared. For the calibration sodium chloride was dried 2 h at 120 °C and 5.845 g were weighed and filled up with water to 1 L.For measuring the release kinetics of Na-4; 0.5*10-4; 1*10-3; 0.5*10-3; 1*10-2; 0.5*10-2 and 1*10-1 mol/L. The measurement was accomplished with normal sensitivity and in the automatic measuring mode. For measuring the release from the controlled-release discs, 70 ml ISA were employed for each disc. After the experiment the measured controlled-release disc was dried and reweighed for control. In this work controlled-release discs with a diameter (D) of 15 mm and a height (H) of 1.1 mm with a sodium carbonate content of 30% (w/w) were tested.For the calibration comparable conditions such as in the biological experiments were applied . The calibration was done at pH values from 3 to 8 and with sodium carbonate concentrations in the applied concentration range in the experiments of 1*10E. coli strains were cultivated in parallel in these flasks under the same conditions as the cultivations in the RAMOS device. For the first sample, depending on the particular experiment, 1 mL or 2 mL of the cultivation broth was withdrawn from the respective flask and the flask was refilled with sterile, purified water. Then, the same respective flask was used for one additional second sample. Each flask was only applied for two samples.To measure off-line data of the experiments, additional Erlenmeyer flasks were used for sampling. The investigated 600) with an Uvikon 922 spectrophotometer , except for the experiment depicted in Fig. seven Thermo Scientific Genesys 20 spectrophotometer  was used. The samples were diluted with fresh medium to measure the OD in the linear range of the photometer and were at least determined twice.Optical densities were measured at 600 nm  and with a Titroline alpha device . For online measurement of the pH during the cultivations, a combination of a RAMOS device and a fiber optical pH-measuring device , as described by Scheidle et al., was used . With thGlycerol, glucose and acetate concentrations were measured with a Dionex HPLC  with an Organic Acid-Resin 250 × 8 mm  and a Skodex RI-71 detector. Sulphuric acid in a concentration of 5 mM was used as solvent at a flow rate of 0.6 ml/min and a temperature of 60°C.E. coli demonstrated that these discs have the most suitable release kinetics for the cultivations (data not shown). The pH-course of the 30% sodium carbonate containing controlled-release disc depicts a fast increase during the first 2 h. Then the pH-value increase nearly linear to a value of about 8.4 after 24 h. The rapid release and pH increase at the beginning of the experiment had to be considered in all biological experiments, because microorganisms tend to show lag phases and initially grow slow and, thus, only few protons are produced. Figure 2O for 30 min to reduce the initial, fast sodium carbonate release. This method was used for some of the cultivation experiments.In Figure E coli BL21 pRSET eYFP-IL6, cultivations with different MOPS buffer concentrations were conducted. Another main focus of these cultivations was to establish a basis for the following experiments using the controlled-release discs to reduce the used buffer concentrations  (from 0.44 1/h to 0.53 1/h) between the cultivations with 0.2 M, 0.1 M and the lower MOPS buffer concentrations can be attributed to the higher osmolarity in the medium with 0.2 M MOPS buffer. The Wilms-MOPS media with 0.2 M and 0.1 M MOPS buffer concentration have an osmolarity of ca. 0.65 Osmol/L and ca. 0.48 Osmol/L, respectively. Record et al. described a linear decrease of the specific growth rate with increasing osmolarities of the medium above 0.3 Osmol/L [The cultivation with 0.1 M MOPS buffer peaked about one hour earlier, than the culture with 0.2 M MOPS buffer concentration. A short oxygen limitation can than be recognized before the OTR decreased slowly in a triangle-shaped form. This triangle-shaped form indicates acidic pH-values that impair the metabolic activity of the microorganisms  see Fig. Furthermax as the cultivation with 0.1 M MOPS buffer until 4.5 h. Here, the OTR decreased earlier in a triangle-shaped form. The low buffer concentration leads to a fast acidification of the medium to a final pH-value of 3.99 and, therefore, to impaired metabolic activity.Application of 0.05 M MOPS demonstrates the same OTR curve and μmax as the culture with 0.1 M MOPS buffer until after 3.5 h the OTR peaks and the OTR slightly decreases. Without buffer, the culture acidifies even earlier and the growth is hampered due to the suboptimal final pH-value of 3.57.The cultivation without any buffer shows the same metabolic activity and μE. coil. However, without the high buffer concentrations the pH decreased very fast to unfavorable values and another method for pH-control is necessary.These cultivations clearly demonstrate the positive influence of reduced buffer concentrations on the specific growth rates of E coli BL21 pRSET eYFP-IL6 in media with different MOPS buffer concentrations and, in addition, controlled-release discs were performed  sodium carbonate content) was performed Figure . Here, tDue to the continuously released sodium carbonate  must be adapted to the particular microorganisms and used cultivation system.With this technique it was possible to halve the buffer concentration in the Wilms-MOPS medium with 20 g/L glucose and to establish the same growth behavior such as that with the higher reference buffer concentration (0.2 M MOPS buffer) at higher grows rates due to decreased osmotic pressure.E coli BL21 pRSET eYFP-IL6 was cultivated in a RAMOS device with integrated online pH measurement [For validating this successful application of the controlled-release discs and to measure the whole pH-course in full detail, surement . One ferE. coli BL21 pRSET eYFP-IL6 cultures with 20 g/L glucose in Wilms-MOPS medium. The pH-value was not kept at one defined value as, e.g. in an actively pH-controlled fermenter. However, the changes in the pH do not exceed 0.6 pH-units and are retained in the optimal pH-range for the metabolic activity of E. coli. Devey specified this optimal range between 6.5 and 7.5 [The pH of the culture with 0.1 M MOPS buffer and three controlled-release discs rose in the beginning of the experiment to a maximum value of ca. 7.7 due to the constant release of sodium carbonate . Therefore, all experiments with glycerol as carbon source used controlled-release discs, which were washed in water for 30 min. During these 30 min, the very fast release of the Na2CO3 in the beginning of the release kinetics can be intercepted. The release kinetics of the washed controlled-release discs are described in Figure Previous experiments with glycerol as carbon source revealed that the growth rate of a-value of MOPS buffer at 37°C is 6.98 and the pH buffer capacity lies in the range of about ±1 of this pKa-value. Therefore, the experiment with initial pH-value of 7.5 can utilize more of the buffer capacity during the cultivation, than the culture with an initial pH-value of 7.The culture with an initial pH 7 and in the absence of controlled-release discs (squares) showed a triangular OTR-curve indicating that the pH-value became too acidic for normal metabolic activity  have to be chosen very carefully to benefit from the pH-control with the polymer-based controlled-release discs. Without the optimal parameters, the pH could drift very fast into too high or too low values for normal metabolic activity of the microorganisms.Using the controlled-release discs at a MOPS buffer concentration of 0.1 M, the culture at an initial pH 7 (circles) depicted a much less impaired metabolic activity compared to the respective culture without sodium carbonate release (squares). Only between ca. 7.5 and 9 h a slightly reduced metabolic activity can be seen in the OTR. The usage of an initial pH of 7.5 and addition of controlled-release discs (triangles) resulted in a preferred OTR curve in this experiment. No impact of suboptimal pH was recognizable. Therefore, an initial pH value of 7.5 is more suitable for To prove the applicability of the controlled-release discs without any addition of buffer in the medium, experiments were performed in Wilms-MOPS medium with 20 g/L glycerol with and without MOPS buffer and controlled-release discs, respectively. A suboptimal initial pH-value of 7  on glycerol of these four experiments in a range of ca. 4.5 and 7.4. Therefore, the controlled-release discs enable the user to control the pH without any additional buffer and the osmolarity of the medium was dramatically reduced from 0.78 OsM in the medium with 0.2 M MOPS buffer to 0.44 OsM in the medium without MOPS buffer. The observed changes in the pH-curve are not ideal for growth of E. coli K12. However, the controlled-release system enabled metabolic activity and complete consumption of the carbon source even without any buffer.In the fourth experiment no buffer and 4 controlled-release discs were investigated Figure . In thisE. coli K12 and E. coli BL21 pRSET eYFP-IL6 in mineral media with glycerol and glucose as carbon sources, respectively. With the controlled-release discs it is possible to substantially reduce the buffer concentrations or even to omit the buffer in media for shake flask cultures, while the pH-values remain in the physiological range for sustained microbial activity during the whole cultivation. This reduction in buffer concentration leads to reduced osmolarities in the medium. These reduced osmolarities may enhance the growth rates of E. coli dramatically, as demonstrated in Figure The presented polymer-based controlled-release discs embedding sodium carbonate crystals for controlling the pH in shake flasks enabled the successful cultivation of Large-scale fermentation processes are equipped with an active pH-control. In these processes no buffers are used. The here presented polymer-based controlled-release discs enable comparable cultivation parameters in shake flasks such as in large-scale, while the buffer concentrations are dramatically reduced and the pH is controlled in a narrow range.In mineral media some of the nutrients, e.g. the phosphate source, function as a buffer. Even in media without any additional buffers, the phosphate concentrations necessary for growth in the media result in a very low buffer capacity. Therefore, when omitting additional buffers while using the controlled-release discs, the medium still has a low buffer capacity. This low buffer capacity can maintain the pH-value to a small degree.2CO3 containing polymer-based controlled-release discs presented in this paper is not self-regulating. Here, the Na2CO3 salt is released and influences the pH-value of the medium, no matter if the microorganisms are growing or not. Additionally, the sodium carbonate is not consumed by the microorganisms such as the glucose in the fed-batch system. Moreover, the nearly linear release of sodium carbonate from the controlled-release discs and the exponential growth leads to inevitable changes in the pH. Therefore, it is very important to ensure optimal growth parameters such as initial pH-values and biomass concentrations while using the newly developed controlled-release discs with pre-defined release kinetics. Especially, the lag phase of a culture has a tremendous effect on the performance of the discs. For example, when the lag phase is too long, the pH increases very fast to suboptimal high values due to the high initial release of the Na2CO3.The controlled-release system for fed-batch cultivation in shaken bioreactors developed by Jeude et al.  is a selThe combination of the controlled-release discs presented in this paper and the glucose delivering controlled-release discs for fed-batch cultivation, described by Jeude et al. , has the2CO3, in response to reducing pH-values are currently under development. This will lead to balanced pH-regulation in small-scale cultures in the future. With such a pH-responsive controlled release system, it will be possible to cultivate microorganisms showing different lag-times in small-scale shaken bioreactors without any additional buffer, thereby ensuring culture conditions in the optimal pH-range.In our Laboratory controlled-release systems which enhance the release of alkaline compounds, such as NaE. coli. DK supervised the preparation of the polymer materials and assisted with data analysis. JB initiated the project, assisted with data analysis and manuscript preparation. All authors approved the final manuscript.MS performed most of the cultivation experiments and prepared the manuscript. BD developed the polymer-based controlled-release discs and measured the release kinetics of the sodium carbonate. JK supported some of the cultivation experiments. HI contributed the cultivation experiment investigating the influence of different buffer concentration on the growth of